Method and application of overexpressing zwf2 gene to increase daptomycin production

A daptomycin, overexpression technology, applied in the field of genetic engineering and microbial fermentation, can solve the problems of low daptomycin production and high production cost, and achieve the effect of reducing production cost and increasing production

Inactive Publication Date: 2011-12-21
TIANJIN UNIV
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Problems solved by technology

Although domestic strains producing daptomycin have already been produced in order to initially realize pilot-scale production, the strains are primitive...

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  • Method and application of overexpressing zwf2 gene to increase daptomycin production
  • Method and application of overexpressing zwf2 gene to increase daptomycin production
  • Method and application of overexpressing zwf2 gene to increase daptomycin production

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Embodiment 1

[0028] Construction of Genetic Engineering Bacteria HP-2 (CGMCC 4132)

[0029] Design primers for PCR clone zwf 2 . (Upstream and downstream primers are respectively: Upper primer: GAGGGCTG CATATG TTGTCTGCTGTTCCC (the underline is the NdeI restriction site); Citation: GTACAA TCTAGA TCACTTGACGCTCCCTG (XbaI restriction site is underlined). The PCR product and the expression vector pIB139 were digested with NdeI / XbaI respectively, and after recovery, the zwf 2 and pIB139 at a ratio of 3:1 to 1:1 were ligated with T4 ligase at 16°C for 2 hours to construct the recombinant expression vector pIB139-zwf 2 (Such as figure 2 ), and then introduce ET12567 Escherichia coli competent cells by the heat shock method, and apply them to the apramycin selection plate after being activated and cultivated in LB medium for one hour. Then the transformants were picked to extract the plasmid for strict verification. Then transfer the recombinant expression vector pIB139-zwf 2 Introduce S...

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Abstract

The present invention provides a method for overexpressing the zwf2 gene to increase the yield of daptomycin and its application, and clones a key enzyme 6-phosphate glucose dehydrogenase gene (zwf2) in the primary metabolic pentose phosphate pathway, using Escherichia coli-Streptomyces Shuttle plasmid expression vector construction of recombinant plasmids. The recombinant plasmid was introduced into Escherichia coli ET12567, ET12567 was co-cultured with Streptomyces roseospora, and Streptomyces roseospora was transformed by the combined transfer method, and then the transformants were screened by apramycin resistance, and the genetically engineered bacteria were further verified by PCR , thereby constructing the genetically engineered strain HP-2. The Streptomyces roseospora genetically engineered bacterium HP-2 constructed by the present invention has an 18% higher output than the starting strain, and the output of daptomycin is as high as 700mg/L, which can be directly used in the industrialized production of daptomycin, which can improve Yield reduces cost.

Description

technical field [0001] The technical field of genetic engineering and microbial fermentation of the present invention, in particular to a kind of overexpression zwf 2 A method for genetically increasing the production of daptomycin and its application. Background technique [0002] Daptomycin ( figure 1 ) is a calcium ion-dependent antibiotic. In the presence of calcium ions, daptomycin will bind to cell membrane proteins in the form of non-covalent bonds. The daptomycin-binding proteins (DBPs) on the cell membrane are Its target site, daptomycin can disrupt the translocation of amino acids by the cell membrane, thereby hindering the biosynthesis of the teichoic acid lipid (LTA) of the bacterial cell wall peptidoglycan and changing the properties of the plasma membrane; Bacteria's cell membrane, so that its contents leak out to achieve the purpose of killing bacteria. It is also reported that its combination with the cell membrane leads to a decrease in membrane potential...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/76C12N1/21C12P21/02C12R1/465
Inventor 闻建平宇光海贾晓强
Owner TIANJIN UNIV
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